Rescue of minireplicon by using the cell line stably expressing the T7 RNA polymerase
Xiu, M-hong.; Wang, Q.; Tang, L-hua.; Cao, S-chun.; Li, W-hong.; Wei, Y.; Lu, P.; Liang, M-fang.; Li, D-xin.
Bing du Xue Bao 23(4): 326-330
2007
ISSN/ISBN: 1000-8721 PMID: 17894237 Document Number: 608490
To improve the rescue efficiency of measles virus cDNA clone, the cell line that stably expressed the T7 RNA polymerase was established. Firstly, the T7 RNA polymerase gene was amplified by PCR and then the PCR product was inserted into pcDNA3 to obtain plasmid pcDNA3-T7. Vero cell was transfected with the plasmid and G418 was added to the cell 24h later to kill the cells without the plasmid. Western blotting analysis showed that the Vero/pcDNA3-T7 cell could express T7 RNA polymerase. To analyze the gene function of T7 RNA polymerase, the pT71P-EGFP plasmid was transfected into the Vero/pcD-NA3-T7 cell and EGFP was analized by fluorescence. The result suggested that T7 RNA polymerase expressed in the Vero/pcDNA3-T7 cell could transcribe the gene under control of the T7 promoter. Moreover. the minigenome PminiEGFP inserted reversely with report gene EGFP was established. After transfection with the plasmid and infection with measles virus, EGFP was expressed, indicating the Vero/pcDNA3-T7 cell could rescue the minigenome.